ABSTRACT: Hematopoietic stem cell transplantation (HCT) is a life-saving therapy, but one that is still plagued with complications, the most deadly of which is graft-versus-host disease (GVHD). Acute GVHD (AGVHD) occurs in as many as 70% of transplant recipients, despite their treatment with multiple immunosuppressive drugs. Moreover, Grade III-IV GVHD, especially involving the GI system, is often untreatable, leading to high rates of post-HCT morbidity and mortality. These issues give rise to three central challenges in the field. They are: (1) What are the mechanisms that drive breakthrough T cell allo-immunity and tissue damage despite current immune suppression strategies? (2) Can we design treatment strategies to directly target these mechanisms? and (3) What are the necessary components of a GVHD-prevention strategy that will safely produce long-term immune tolerance? To address these questions, we have developed and refined the only non-human primate (NHP) model of GVHD, and have used this model to discover a series of new insights into the immunology of this disease. These include: (1) That a systems biology approach can be applied to GVHD to uncover central mechanisms and targetable pathways. (2) That AGVHD can be divided into ?primary? and ?breakthrough? mechanisms: with primary GVHD driven by Th/Tc1 pathways, while breakthrough GVHD is driven by IL17- predominant pathways. (3) That primary AGVHD can be successfully controlled by calcineurin Inhibitor-free CD28:CD80/86 blockade + rapamycin. These discoveries form the core of our new understanding of AGVHD and inform the next phase of our work. This work is based on the overarching hypothesis that the tissue- specific molecular mechanisms controlling GVHD can be identified, and that by targeting these mechanisms, an evidence-based approach to the prevention and treatment of this disease can be achieved. We will test this hypothesis through the following Specific Aims: Aim 1: Evidence-based GVHD Prevention: This Aim will determine strategies by which immune escape pathways, identified through transcriptome analysis, can be targeted to prevent GVHD. Aim 2: Tissue-Specific GVHD Diagnostics: In this Aim, we will establish a transcriptomic map comparing blood- liver, and GI-specific immune activation during NHP GVHD, using both population- and single-cell techniques. Aim 3: Evidence-based GVHD Treatment: In this Aim we will determine the mechanisms controlling breakthrough GVHD in both NHP and patients and test novel treatment strategies in our newly-developed NHP GVHD Treatment Model.